The Mechanisms Of Rat’s Cognitive Deficits Induced By Melamine And The Potential Treatment Effect Of VC And VE Joining Application

Melamine (1,3,5-Triazine-2,4,6-triamine), a synthetic chemical compound, is often used to produce melamine resin by reacting with formaldehyde. They have been used to manufacture plastics, adhesives and fabrics. Although melamine has been permitted to be used in some food contact products (e.g. food package materials and tableware) for a quite long time, it is not a kind of food additive and is never approved to be added to food directly.The study of melamine toxicity in animals started as early as1984, however, it did not attract widespread public concern. Ample evidence certified that infants and animals were affected by food which were contaminated by melamine, and the renal pathology was the main manifestation in intoxicated case. Our previous studies showed that melamine produced the impairment of hippocampal function and induced apoptosis and oxidative damage in PC12cell. Obviously, the known nephrotoxicity of melamine could not fully explain its neurotoxicity effects.On the other hand, its impact may persist longer than we thought. Antioxidants have been shown to decrease some of the biochemical measures of oxidative stress, and then mitigate function impairment of CNS. Vitamin C and E are two essential nutrients that can scavenge free radicals and constitute a strong line of defense in retarding reactive oxygen species (ROS)-induced cellular damage. Combination of vitamin C and E treatment can significantly attenuate oxidative damage, anxiety disorders and cognitive-behavioral impairment in AD, PD, amyotrophic lateral sclerosis and ataxia and improve their daily life viability as well as social activity.Against above background, we investigated the effects of melamine on the spatial cognition and hippocampal synaptic plasticity of Wistar rats by gavage for28consecutive days. Meanwhile, hippocampal ROS and antioxidative enzyme activities were detected. Subsequently, we explored the antioxidative effect of vitamin C and E combination on melamine-induced neurotoxicology and attempted to make clear the potential mechanism.Part1the effects of melamine on the spatial cognition and hippocampal synaptic plasticity in ratsObjective:To investigate the effects of melamine on spatial cognition and hippocampal synaptic plasticity in Wistar rats, and explore the potential mechanism.Methods:Male adolescent Wistar rats, three-week-old, were randomly divided into two groups:control group (n=16) and melamine group (n=16). Rats were intragastric administered with melamine at a dose of300mg/kg/day once a day for4weeks. After that, Morris water maze (MWM) test was performed to evaluate the spatial cognitive function, including learning, reference memory and re-acquisition abilities. The long term potentiation (LTP) and long-term depression (LTD) from Schaffer collaterals to hippocampal CA1region were measured. In addition, the melamine concentration in the hippocampus was detected by enzyme linked immunosorbent assay (ELISA) method.Results:1. The ELISA test showed that the melamine content in the hippocampus was abnormally increased after melamine-treatment for28days, which indicated that melamine could pass through blood brain barrier (BBB).2. The MWM test showed that the escape latencies in both initial training (IT) and reversal training (RT) stages were significantly increased in melamine group compared to that in control group (P<0.01). In space exploring test (SET) stage, both the platform crossings (P<0.01) and the quadrant dwell time (P<0.01) were decreased in melamine group compared to that in control group.3. LTP recording were showed that the slope of field excitatory postsynaptic potentials (fEPSPs) was significantly lower in melamine group than that of control group (P<0.01). Meanwhile, LTD test was found that the fEPSPs slope was dramatically higher in melamine group than that in control group (P<0.01).Conclusions:Melamine is able to pass the BBB and accumulate in the hippocampus region. Meanwhile, the deleterious effects of melamine on hippocampal long-term synaptic plasticity broke the balance between stability and flexibility of cognition, which could further explain the poor performance in behavioral tests.Part2Spatial cognition was impaired by melamine associated with oxidative damage in ratObjective:To investigate whether cognitive deficits induced by melamine was associated with the impairment of oxidation-antioxidation homeostasis.Methods:Male adolescent Wistar rats, three-week-old, were randomly divided into two groups:control group (n=8) and melamine group (n=8). Rats were intragastric administered with melamine at a dose of300mg/kg/day once a day for4weeks. After that, the MWM experiment and histopathological examination were performed. Meanwhile, the level of reactive oxygen species (ROS), malonaldehyde (MDA) superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and adenosine-triphosphate (ATP) levels were detected in the hippocampus.Results:1. During the four weeks of melamine treatment, rats gained significant less weight in melamine group than those in control group (P<0.01). There was an abnormal increasing in the concentration of melamine in hippocampus in melamine group.2. In IT stage of MWM test, it was significantly prolonged in melamine group compared to that in control group (P<0.01). Furthermore, in SET stage, it was found that both the platform crossings (P<0.01) and the quadrant dwell time (P<0.01) were decreased in melamine group compared to that in control group. However, swimming speeds of these two groups remained constant throughout testing and no difference was found between them (P>0.05).3. Results of biochemical tests:Chronic melamine exposure induced dysfunction of oxidation-antioxidation homeostasis, including increased the levels of ROS and MDA, decreased SOD, GSH-Px and ATP.Conclusions:The neurotoxicity of melamine in hippocampus may be partly associated with oxidative damage. Part3The reversal effects of VC and VE joining application on hippocampal oxidative stress induced by melamine in PC12cellsObjective:To explore the potential treatment efficacy of vitamin C and vitamin E alone, the combination of vitamin C and E on oxidative stress induced by melamine.Methods:PC12cells were cultured with melamine to establish an in vitro model of neuronal cells. The cellular viability was tested by MTT assay. The flow cytometry was used to detect the apoptosis of PC12cells and the level of intracellular reactive oxygen species (ROS). Furthermore, the inverted phase contrast microscope was used to observe the morphology of cells by Hoechst33342staining. The biochemical assays were used to evoluate the effect of antioxidative vitamins on the neurotoxicology of melamine.Results:1. Compared with vitamin C and vitamin E alone, the combination of vitamin C and E was statistically increased PC12cells viability as demonstrated by MTT assay.2. The further evidences indicated that vitamin complex was effectively reduced the formation of reaction oxygen species ROS), decreased the level of MDA and elevated the activities of antioxidative enzymes, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px).3. Hoechst33342staining and flow cytometric analysis of apoptosis revealed the apoptotic nuclear features of the melamine-induced cell death, and showed that vitamin combination, but not vitamin C or vitamin E, effectively prevented PC12cells from this melamine-induced apoptosis.4. The combination of vitamins effectively inhibited apoptosis via blocking the incesased activation of caspase-3.Conclusions:Vitamin E and C combination protect PC12cells from the injury induced by melamine through the down-regulation of oxidative stress and prevention of melamine-induced apoptosis.Part4The potential treatment effects of VC and VE joining application on spatial cognition and its possible mechanisms in melamine-treated ratsObjective:To investigate the antioxidative effect of combination of vitamin C and E on cognitive function in melamine rats and explore the potential mechanism.Methods:Male adolescent Wistar rats, three-week-old, were randomly divided into four groups:control group (n=16), melamine group (n=16), melamine+vtamin group (n=16) and vitamin group (n=16). Rats were intragastric administered with melamine at a dose of300mg/kg/day once a day for4weeks. Rat in melamine+vitamin group received vitamins C and E at a dose of150and200mg/kg respectively, intraperitoneally once a day for the next7consecutive days. After that, spatial cognitive function was investigated using a spatial version of the Morris water maze test. The long term potentiation (LTP) and long-term depression (LTD) from Schaffer collaterals to hippocampal CA1region were recorded. In addition, the levels of ROS, MDA and NO and the activities of SOD, CAT, GSH-Px, NOS, apoptotic related proteins Bcl-2and caspase-3were detected by ELISA method.Results:1. Rats treated with vitamins E and C had part of the above effects prevented in MWM tests, with mitigating the melamine-induced deficit in the learning and memory stage of MWM test but slightly improving the reversal learning ability.2. Histological observation indicated that vitamin complex effectively alleviated the injuries of hippocampal neurons induced by melamine.3. In vivo electrophysiological assessment found that melamine was able to cause the impairment of hippocampal synaptic plasticity drastically. The vitamins C plus E regimen mitigated the melamine-induced impairment of hippocampal synaptic plasticity, with an entirely ameliorated LTP and a significantly reversal LTD. Furthermore, in the S group, this vitamin regimen did not produce obviously deficits in behavioral and electrophysiological experiments.4. ROS, MDA, NO contents were reversed to control levels after treatment with combination of vitamin C and E. SOD, CAT, GSH-Px, NOS activities were also improved compared to melamine-treated group. The neural apoptosis in the hippocampus were ameliorated by regulating the expression of pro-apoptotic protein (Bcl-2) and anti-apoptotic protein (caspase-3). Conclusions:It is concluded that oxidative damage plays an important role in the melamine-induced hippocampal impairment. The modulation of oxidative stress with vitamins E and C reduced the melamine-induced damage and may support to a novel therapeutic strategy to the cognitive dysfunction observed in melamine-induced patient.Part5Spatial cognition and sexually dimorphic synaptic plasticity balance impairment in rats with chronic prenatal ethanol exposureObjective:To investigate the sexual different effects of chronic prenatal ethanol exposure (CPEE) on behavior cognition and cognitive flexibility, and interpret the possible mechanism by evaluating the alternation of synaptic plasticity balance.Methods:The animal model was produced by ethanol exposure throughout gestational period with4g/kg bodyweight. The offspring of both male and female was selected and studied on postnatal days36. After that, the Morris water maze (MWM) test was performed to evaluate the spatial cognitive function, including learning, reference memory and re-acquisition abilities. LTP and depotentiation from Schaffer collaterals to hippocampal CA1region were recorded.Results:1. It demonstrated that chronic ethanol exposure reduced birth weight, losing bodyweight gain, microcephaly and retarded hippocampal weight.2. In Morris water maze (MWM) test, it was found that the latencies were significantly higher in adolescent CPEE-treated rats than that in normal rats. The time spent of CPEE-treated rats was markedly increased near the periphery of the pool. Furthermore, they took less time in the target quadrant and reduced platform crossing times in the probe phase.In addition, the behavioral test also showed that there was a particularly serious impairment of females than that of males after CPEE treatment.3. Electrophysiological studies showed that CPEE dramatically depressed long-term potentiation (LTP) and obviously enhanced depotentiation in male adolescent hippocampus, while drastically exaggerated LTP and hardly impacted depotentiation in female.4. Our original index also displayed that synaptic plasticity balance of female was much more sensitive to the effect of CPEE than that of males.Conclusions:The investigation revealed the sex different effect of CPEE on synaptic plasticity balance and provided a better understanding of CNS dysfunction in CPEE treatment.